Uncovering the Regulatory Role of gH/gL in HSV Fusion

描述（由申请人提供）：单纯疱疹病毒引起人类疾病，从唇疱疹到更严重的感染，其中一些可能危及生命。我们的长期目标是描述控制HSV进入和HSV诱导的细胞融合的机制。与大多数病毒不同，HSV利用多种蛋白质在多步骤过程中融合和进入。这些事件的特征将有可能确定病毒介导融合的新范式。四种HSV糖蛋白，gD, gB， gH和gL是进入必需的。gD是HSV的受体结合蛋白，而gB和gH/gL复合物形成了所有疱疹病毒的核心融合机制。gB、gD和与其受体结合的gD的晶体结构为了解这些蛋白在病毒进入和细胞融合中的功能提供了重要的见解。gB与VSV融合蛋白G的结构同源性表明，gB在所有疱疹病毒中都是一个融合蛋白。然而，与G不同的是，gB不能单独发挥作用，而是需要gH/gL。同样，先前的研究表明gH/gL本身是一个很差的融合原。主要基于序列基序、合成肽和突变的主流模型认为，这两种弱融合原的结合是通过在同一膜（如病毒粒子）中共同作用来促进融合来完成融合的。我们最近与E. Heldwein博士合作，解决了gH/gL的晶体结构。令人惊讶的是，gH/gL具有与任何已知的病毒融合蛋白不同的新结构，强烈反对其作为融合原的作用。这一结构表明，gB是HSV唯一具有内在融合能力的进入蛋白。因此，我们认为gH/gL具有非常不同的作用。我们假设gH/gL通过与gB结合并激活其进入融合状态而发挥融合调节作用。为了支持这一假设，我们发现gH/gL外畴本身（非膜结合）可以触发gD和gB转染的受体承载细胞的融合。在目标1中，我们将剖析gH/gL与gB与突变体和单克隆抗体形成的复合物的性质。诸如生物传感器分析、量热法、低温电子显微镜断层扫描和x射线晶体学等技术将被用于获得有关络合物的化学计量学和构象的信息。双分子荧光互补（BiMC）和融合实验将用于分析gB和gH/gL在同一细胞（顺式）和不同细胞（反式）中的复合物形成。我们将确定突变形式的gH/gL触发gB进入融合状态的能力。在目标2中，我们将通过改变WT和可溶性gH/gL突变形式的速率和浓度等参数来测试gH/gL作为融合调节剂的作用。我们将使用BiMC来跟踪和比较HSV进入时通过质膜直接融合或内吞作用发生的gB-gH/gL复合物的形成。我们在HSV进入和融合方面的工作具有很强的临床意义，因为每一步都是针对HSV介导疾病的治疗和疫苗的潜在靶点。为了验证我们的主要假设，我们提出了两个具体目标：1)进一步表征gH/gL并剖析其与gB形成的复合物的性质；2)确定gH/gL如何激活gB进入融合状态。

DESCRIPTION (provided by applicant): Herpes simplex virus causes human diseases, ranging from cold sores to more serious infections, some of which can be life threatening. Our long term goal has been to delineate the mechanisms that govern HSV entry and HSV induced cell fusion. In contrast to most viruses, HSV utilizes multiple proteins in a multi-step process for fusion and entry. Characterization of these events will likely identify novel paradigms for virus-mediated fusion in general. Four HSV glycoproteins, gD, gB, gH and gL are essential for entry. gD is the receptor binding protein for HSV, while gB and the gH/gL complex form the core fusion machinery for all herpesviruses. Crystal structures of gB, gD and gD bound to its receptors have provided critical insights about how these proteins function in virus entry and cell-fusion. The structural homology of gB with G, the fusion protein of VSV, suggests that gB is a fusion protein in all herpesviruses. Unlike G however, gB does not function on its own but requires gH/gL. Likewise, prior studies showed that gH/gL is a poor fusogen on its own. The prevailing model, based largely on sequence motifs, synthetic peptides and mutations, is that the combination of these two poor fusogens accomplish fusion by acting together in the same membrane (as in virions) to promote fusion. In collaboration with Dr. E. Heldwein, we recently solved the crystal structure of gH/gL. Surprisingly, gH/gL has a novel architecture distinct from any known viral fusion protein, strongly arguing against its role as a fusogen. This structure implies that gB is the only entry protein of HSV that is intrinsically capable of causing fusion. We therefore suggest that gH/gL has a very different role. We postulate that gH/gL functions as a regulator of fusion by binding to gB and activating it into a fusogenic state. In support of this hypothesis, we discovered that the gH/gL ectodomain itself (not membrane bound) can trigger fusion of receptor-bearing cells transfected with gD and gB. In Aim 1, we will dissect the nature of the complex formed by gH/gL with gB with mutants and monoclonal antibodies. Techniques such as biosensor analysis, calorimetry, cryo-EM tomography and X-ray crystallography will be used to gain information about the stoichiometry and conformation of the complex. Bimolecular fluorescence complementation (BiMC) and fusion assays will be used to analyze complex formation when gB and gH/gL are in the same cell (cis) and in different cells (trans). We will determine the ability of mutant forms of gH/gL to trigger gB into a fusogenic state. In Aim 2, we will test gH/gL as a regulator of fusion by varying such parameters as rate and concentration of the WT and mutant forms of soluble gH/gL. We will use BiMC to follow and compare gB-gH/gL complex formation during HSV entry when it occurs by direct fusion at the plasma membrane or by endocytosis. Our work on HSV entry and fusion has strong clinical significance, as each step is a potential target for therapeutics and vaccines against HSV-mediated disease. To test our main hypothesis, we propose two specific aims: 1) to further characterize gH/gL and dissect the nature of the complex it forms with gB; 2) To determine how gH/gL activates gB into a fusogenic state. PUBLIC HEALTH RELEVANCE: Herpes simplex virus (HSV) causes many human diseases but the most common are cold sores and encephalitis. The outer surface of the virus contains the proteins that are needed to gain entry into host cells and cause disease and I use molecular biological approaches to understand how these proteins work to allow the virus to enter host cells. My research may suggest new therapeutic targets that will result in new treatments to stop the virus from gaining entry into its favorite target cells.